The formation of signaling gradients within tissues is a fundamental aspect of animal development. Graded signals function to specify cells with different identities, causing them to follow independent, developmental programs depending on signal concentration. In the developing vertebrate central nervous system, signals from the embryonic mesoderm pattern the primary axes of an apparently homogeneous neuroepithelium, specifying different neuronal types and their interconnections to form a functional neural network. In the hindbrain in particular, this translates into boundaries between rhombomeres, producing a series of 7 segments along the anterior-posterior (A-P) axis. Surprisingly little is known, however, about the molecular mechanisms that control early A-P patterning in vertebrates and we are addressing this issue using the genetic and embryological advantages of zebrafish. In the hindbrain, signaling mediated by the vitamin A derivative, retinoic acid (RA) and its nuclear receptors (RARs) has been implicated in neural patterning, but our preliminary studies show that a simple A-P gradient of RA signaling is insufficient to explain the entire process. The long-term goal of the proposed research is to understand the cellular and molecular basis of A-P patterning during vertebrate hindbrain development. 2 primary hypotheses guide the proposed research. First, we hypothesize that local degradation of RA by enzymes of the cyp26 family is the key to its graded effects on the hindbrain. Secondly, we hypothesize that restricted domains of RAR expression determine tissue-specific responses to RA in the hindbrain and in adjacent cranial mesoderm. Aim 1 is to follow the response to RA in living embryos using an RA-response element driving GFP, to define the range and concentration-dependence on RA in the hindbrain. The optical clarity and availability of transgenic zebrafish make it uniquely suited for this study. Aim 2 is to analyze the functions of RA degradation on signaling, by disrupting the endogenous expression of cyp26s and/or their autoregulation by RA itself. Aim 3 focuses on the roles of individual RARs using a combination of mutants, morpholinos and pharmacological antagonists uniquely available in zebrafish. Here we will focus initially on a particular tissue interaction that requires RARg, between the mesoderm and developing neural tube, and will use cell transplantation in RAR-deficient embryos to get to the biochemical basis for neural patterning. [unreadable] [unreadable] [unreadable]